Insect cell primary culture medium, extracellular matrix, and process of preparing an insect culture cell line in a short period of time using the medium and matrix

ABSTRACT

A novel cell culture medium suitable for primary culture of insect cells, an insect-derived water-soluble chitin, and a process of preparing an insect culture cell line in a short period of time by using the insect primary culture medium and the insect-derived water-soluble chitin. The insect cell primary culture medium comprises lactalbumin hydrolysate, yeastolate, and tryptose phosphate broth as protein extracts, and polyvinylpyrrolidone as a viscosity-supplementing agent. The insect-derived water-soluble chitin is subjected to deacetylation as the sole chemical modification.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a technique for preparing an insectculture cell system.

2. Prior Art

There are two stages in the preparation of an animal culture cell lineincluding that of insects. The initial stage is called primary culture,and the next stage is called subculture. When a culture cell line isprepared, a target tissue is extracted from an insect of interest in anaseptic manner. The tissue is then put into a culturing flask togetherwith a cell culture medium, and cultured for about one year, until cellsemerge from the tissue section onto the flask surface and divide intosufficient numbers. The sufficiently multiplied cells are transplantedinto a new flask where they are subjected to subculture, i.e., culturingof a prepared cell line.

In the prior art, a variety of insect cell media are available. Examplesinclude Grace's media, IPL-41 media, Schneider's Drosophila media,Sf900II, TC-100 media, Sf-9 cell media, Sf-21 cell media, Express Fivemedia, and EX-400 media. Mitsuhashi, J. et al. have prepared a mediumalong the line of MGM, for example, by referring to the results ofinsect body fluid analysis (MGM-443; Mitsuhashi, J. (1980) In: Kurstak,E., Maramorosch, K. and Dubendorfer, A. (eds) Invertebrate Systems InVitro Elsevier/North Holland Biomedical, Amsterdam, pp. 47–58, MGM-448;Mitsuhashi, J. (1984) Zool. Sci. 1, pp. 415–419, MGM-450; Mitsuhashi, J.and Inoue, H (1988) Appl. Entomol. Zool. 23, pp. 488–490, MGM-464;Mitsuhashi, J. (2001) In Vitro Cell. Dev. Biol. 37A, pp. 330–337.MGM-443, MGM-448, MGM-450, and MGM-464 are media names.).

The present inventors have prepared MM-8 SF medium, for example (ShigeoImanishi, “Konchu Kino Jikken-kei Oyobi Konchu Saibo Baiyo-kei NoKaihatsu: Kenkyu Seika 295” (“Development of Insect Function -ExperimentSystem and Insect Cell Culture System: Study Results 295”), ed. byAgriculture, Forestry, and Fisheries Research Council, pp. 74–84(1994)). These media, however, are not suitable for primary culture, andare used mostly for the subcultivation of a prepared culture cell line,i.e., for the maintenance of the cells after development of the cellline. When these media were used for primary culture, cell growth wasnot satisfactory, and primary culture took as long as about one year, asmentioned above. Thus, there has been no medium suitable for primaryculture which enables the cells to be transferred into subculture at anearly stage. Moreover, many of these media were limited for use withcertain insect species. For example, the Schneider's Drosophila mediumwas for the culturing of cells of insects of the order Diptera,particularly fruit flies, while Sf900II, Sf-9 cell medium and Sf-21 cellmedium were for the cells of insects of the order Lepidoptera,particularly inch worms. Thus, there has so far been no medium suitablefor the growth of cells of insects of a wide variety of orders.

On the other hand, flasks generally used for cell culture are made ofplastic, and are believed to have a coating of extracellular matrix onthe plastic surface, such as, e.g., collagen I, II, III, IV, or V,fibronectin, gelatin, laminin, poly-L-lysine, Matrigel, and EHS-matrix.There are not many detailed reports about their compositions, but as tochitin and chitosan, there have been reports about an extracellularmatrix extracted from crustaceans such as lobsters and crabs. Thesechitin and chitosan have been variously chemically modified. Theextracellular matrix provides the effect of attaching a tissue to thecell culture vessel, and adhering those cells that have transmigratedfrom the tissue to the vessel surface for division and multiplication.However, there is a need for an extracellular matrix with a higher celladhesion ability for good cell culture.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a novelcell culture medium suitable for primary culture of insect cells.Another object of the present invention is to provide an insect-derived,water-soluble chitin that has been subjected to deacetylation as theonly chemical modification. Yet another object of the present inventionis to provide a process of preparing an insect culture cell line in ashort period of time which utilizes the above insect cell primaryculture medium and insect-derived, water-soluble chitin.

As described above, there is a need for a medium suitable for primaryculture of insect cells, i.e., an insect cell medium which facilitatesthe division and multiplication of only those cells (primary cells) thatemerged on the bottom surface of the culture flask from the slicedtissue segment after start of cell culture, thereby enabling the cellsto be transferred to subculture at an early stage.

After careful examination and analysis of media suitable for primaryculture which facilitate the division and multiplication of cellsobtained from a tissue fragment and which enable the cells to betransferred to subculture at an early stage, the inventors arrived atthe present invention after realizing that a novel medium containingcertain protein extracts and a viscosity-supplementing agent wassuitable for primary culture.

The inventors also carefully examined and analyzed a novel extracellularmatrix to be coated on a culture vessel which is suitable for culturinginsect cells. As a result, the inventors arrived at the presentinvention after finding that when cells are cultured in a cell culturevessel coated with an extracellular matrix comprising an insect-derived,water-soluble chitin that has not been subjected to chemicalmodification other than deacetylation, the attachment of the tissue ontothe culture vessel can be improved and that cells that havetransmigrated from the tissue also adhere to the vessel surface anddivide and multiply. Furthermore, the inventors arrived at the presentinvention after finding that primary culture can be performed moreefficiently by using a cell culture vessel which is coated with anextracellular matrix comprising an insect-derived, water-soluble chitinthat has been subjected to no chemical modification, and a mediumsuitable for primary culture.

Namely, the present invention provides the following:

(1) an insect cell primary culture medium comprising lactalbuminhydrolysate, yeastolate, and tryptose phosphate broth as proteinextracts, and polyvinylpyrrolidone as a viscosity-supplementing agent;

(2) an insect cell primary culture medium (1) comprising 1000–3000 mg/Lof lactalbumin hydrolysate, 1000–3000 mg/L of yeastolate, 1000–3000 mg/Lof tryptose phosphate broth, and 200–500 mg/L of polyvinylpyrrolidone;

(3) an insect cell primary culture medium (1) or (2), wherein thepolyvinylpyrrolidone is polyvinylpyrrolidone K-90;

(4) an insect-derived, water-soluble chitin that has been subjected todeacetylation as the sole chemical modification;

(5) an insect-derived, water-soluble chitin (4), wherein the chitin hasbeen derived from silkworm;

(6) an insect-derived, water-soluble chitin (5), wherein the chitin hasbeen derived from silkworm pupa exuvia;

(7) an extracellular matrix comprising any one of the insect-derived,water-soluble chitin (4) to (6);

(8) an extracellular matrix solution for coating a culture vessel,comprising 0.001% to 1% of any one of the insect-derived, water-solublechitin (4) to (6);

(9) an insect cell culture vessel coated with any one of theinsect-derived, water-soluble chitin (4) to (6);

(10) a process of manufacturing the insect-derived, water-soluble chitin(6), which comprises extracting chitin from a silkworm pupa exuvia anddeacetylating the chitin;

(11) a process of preparing an insect culture cell line in a shortperiod of time, which uses any one of the insect primary culture cells(1) to (3), and any one of the insect-derived water-soluble chitin (4)to (6); and

(12) a process of preparing an insect culture cell line in a shortperiod of time according to (11), wherein a vessel coated with any oneof the insect-derived water-soluble chitin (4) to (6) is used, and aninsect cell is cultured on any one of the insect primary culture media(1) to (3).

DESCRIPTION OF THE INVENTION

The present invention will be hereinafter described in detail.

1. Composition of the Insect Cell Primary Culture Medium According tothe Invention

The insect cell primary culture medium according to the presentinvention comprises at least a protein extract and aviscosity-supplementing agent. The primary culture medium according tothe present invention may further comprise a mixed composition ofinorganic salts, a sugar composition, a mixed composition of aminoacids, and a mixed composition of vitamins.

Examples of the protein extract include at least lactalbuminhydrolysate, yeastolate, and tryptose phosphate broth, and may furtherinclude fetuin, cytochrome c, inosine, bovine plasma albumin V, etc.Lactalbumin, yeastolate, and tryptose phosphate broth may be obtained ina known manner. Fetuin may be derived from fetal calf serum, forexample, while cytochrome c may be derived from horse heart, forexample. These protein extracts may all be commercially available ones.For example, lactalbumin hydrolysate may be from Difco (No. 5996), andyeastolate may be prepared for cell culture (TC(tissue culture)yeastolate), such as from Difco (No. 5577), and tryptose phosphate brothmay be from Difco (No.0060). Further, fetuin may be from Sigma (No.F2379), cytochrome c may be from Sigma (No. C2506), and inosine may befrom Wako Pure Chemical Industries, Ltd.

The individual content of lactalbumin hydrolysate, yeastolate, andtryptose phosphate broth in the medium should preferably be in the rangeof from 500 to 3000 mg per 1 L of the medium, more preferably in therange of from 1000 to 3000 mg, and most preferably in the range of from1000 to 2000 mg. The content of fetuin should preferably be in the rangeof from 1 to 100 mg per 1 L of the medium, more preferably in the rangeof from 1 to 50 mg, and most preferably in the range of from 5 to 15 mg.The content of cytochrome c should preferably be in the range of from 1to 500 mg per 1 L of the medium, more preferably in the range of from 1to 100 mg, and most preferably in the range of from 10 to 100 mg. Thecontent of inosine should preferably be in the range of from 1 to 500mg, more preferably be in the range of from 1 to 200 mg, and mostpreferably be in the range of from 10 to 200 mg. The content of bovineplasma albumin V should preferably be in the range of from 1000 to 10000mg, more preferably be in the range of from 100 to 10000 mg, and mostpreferably be in the range of from 1000 to 10000 mg.

As the viscosity-supplementing agent contained in the primary cellculture medium according to the present invention, polyvinylpyrrolidoneis used. Examples of polyvinylpyrrolidone include, e.g.,polyvinylpyrrolidone K-25, polyvinylpyrrolidone K-30, andpolyvinylpyrrolidone K-90. Though any of these may be used,polyvinylpyrrolidone K-90, such as, e.g., one manufactured by Wako PureChemical Industries, Ltd. (their catalog No. 168-03115), is preferablyused. The content of polyvinylpyrrolidone should preferably be in therange of from 100 to 1000 mg per 1 L of the medium, more preferably bein the range of from 100 to 500 mg, and most preferably be in the rangeof from 200 to 500 mg.

The mixed composition of inorganic salts, sugar composition, mixedcomposition of amino acids, and mixed composition of vitamins that areadded may be ones that can generally be added in an animal cell medium.The mixed composition of inorganic salts may contain NaH₂PO₄, NaHCO₃,KCl, CaCl₂, CuCl₂, CoCl₂, FeSO₄, MgCl₂, MgSO₄, MnCl₂, NaCl, NaH₂PO₄,(NH₄)₆(Mo₇O₂₄.4H₂O), and ZnCl₂. The sugar composition may containglucose, fructose, sucrose, malic acid, α-ketoglutaric acid, succinicacid, fumaric acid, and maltose. The mixed composition of amino acidsmay contain α-alanine, β-alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine,leucine, hydroxyproline, lysine, methionine, phenylalanine, proline,serine, threonine, tryptophan, tyrosine, and valine. The mixedcomposition of vitamins may contain biotin, D-calcium pantothenate,choline chloride, folic acid, i-inositol, nicotinic acid, pyridoxine,riboflavin, thiamin, vitamin B₁₂, para-aminobenzoic acid. Preferably,the mixed composition of inorganic salts, sugar composition, mixedcomposition of amino acids, and mixed composition of vitamins shouldcontain all of the above substances. They may, however, lack some of theabove substances, or be added with other substances. They may all becommercially available ones. There may also be used a commerciallyavailable inorganic salt composition, sugar composition, mixedcomposition of amino acids, and vitamin composition for addition intomedia. Alternatively, the above-mentioned protein extracts andviscosity-supplementing agent may be added to a known medium containinginorganic salts, sugars, amino acids and vitamins as principalconstituents. In this case, examples of the known medium include knowninsect cell culture media such as Grace's medium and Schneider'sDrosophila medium. Further, antibiotics such as penicillin andstreptomycin, and glutathione may be added to the medium. In otherwords, the medium according to the present invention includes all themedia that contain at least lactalbumin hydrolysate, yeastolate,tryptose phosphate broth and polyvinylpyrrolidone.

When the medium according to the present invention is used forculturing, it is preferable to add animal serum such as, e.g., fetalbovine serum, or insect lymph, for example. The amount of the serum orlymph added is in the range of from a few % to 30%.

The insect cell primary culture medium according to the presentinvention can be obtained by mixing medium additives comprisinglactalbumin hydrolysate, yeastolate, tryptose phosphate broth andpolyvinylpyrrolidone, with other protein extract mixed compositions,mixed compositions of inorganic salts, sugar compositions, mixedcompositions of amino acids, and mixed compositions of vitamins.Accordingly, a medium additive comprising at least lactalbuminhydrolysate, yeastolate, tryptose phosphate broth andpolyvinylpyrrolidone is included in the scope of the present invention.

One example of the primary cell culture medium according to the presentinvention is MX medium.

The primary cell culture medium according to the present invention canbe prepared by dissolving in pure water the above-mentioned proteinextracts, viscosity-supplementing agent, inorganic salt mixed compounds,sugar compounds, amino acid mixed compounds, and vitamin mixedcompounds. There is further added a required amount of theabove-mentioned animal serum or insect lymph. If necessary, pH isadjusted with an acidic solution such as, e.g., hydrochloric acid, or abasic solution such as, e.g., sodium hydroxide. The pH should preferablybe in the range of from 6.0 to 7.0, most preferably in the range of from6.2 to 6.5.

2. Use of the Medium According to the Invention for Primary Cell Culture

The medium according to the present invention can be used for primaryculture of any and all insect cells. It is particularly suitable forinsects of the orders Lepidoptera, Diptera, Coleoptera, and Hemiptera,for example. Further, a culture cell line can be created in a shortperiod of time from any and every tissue of these insects. Particularly,the inventive medium is suitable for culturing from embryonic tissue,fat body tissue, reproductive tissue of testis or ovary, digestivesystem tissue, nervous system tissue, and muscular system tissue. Thus,with the medium according to the present invention, it is possible tocreate a culture cell line in a short period of time even from thetissue of testis, for example, from which creation of a culture cellline has formerly been believed difficult.

A tissue from which a culture cell line is desired to be created isextracted from an insect in an aseptic manner, and then put into a cellculture vessel such as a cell culture flask, together with the mediumaccording to the present invention. The tissue is cultured for two tothree months while exchanging or supplementing the medium as needed. Inthis way, multiplied cell populations can be transplanted into a newflask in a shorter period of time than possible in the prior art. Inthis case, culture conditions may be those for conventional insect cellculture. For example, the cells may be cultured in an incubator attemperatures in the range of from 20 to 28° C. The created culture cellline can be cultured after being transferred to a known subculturemedium such as, e.g., MGM-464 medium, IPL-41 medium, Grace's medium,EXCELL 400-line medium, Sf900II medium, Schneider medium, etc. Thus,compared with conventional media, a faster transfer to subculture can beachieved with the medium according to the present invention.Alternatively, the cells may be cultured continuously on the mediumaccording to the present invention. Even if the proportion of the serumcontent added to the medium of the present invention is decreased, thecells can nevertheless divide and multiply well, so the cells can besubcultured over a long period of time.

3. Extracellular Matrix According to the Invention

In the present specification, the term “extracellular matrix” refers toa matrix, substrate or carrier to which cells can adhere and divide andmultiply during cell culture. Preferably, the extracellular matrix isused for coating the cell culture surface of the culture vessel.

The extracellular matrix according to the present invention comprises asa principal constituent, an insect-derived, water-soluble chitin thathas been subjected to deacetylation as the sole chemical modification.

The water-soluble chitin may be derived from any insect. Preferably, itis extracted from exuviae of the pupa of an insect, particularlysilkworm.

The water-soluble chitin according to the present invention can beobtained by the following manner.

A 1 N solution of hydrochloric acid is poured on silkworm pupa exuviae.The exuvia is treated in an environment filled with nitrogen gas at 100°C. for 20 min. Then, protein is removed in a 1 N solution of sodiumhydroxide at 80° C. over a period of 36 hours, thereby preparing chitin.To prepare a water-soluble chitin, the chitin is dissolved in aconcentrated alkali water solution at room temperature, and thehigh-viscosity alkaline chitin water solution is allowed to stand atroom temperature for a long time to thereby deacetylate in a randommanner. Water solubility is exhibited only when the degree ofdeacetylation is in the range of from 45 to 55%.

For the preparation of the water-soluble chitin for the extracellularmatrix according to the present invention, preferably a chitin with highaffinity for water should be selected. The affinity of chitin for watercan be evaluated according to the method of Brunauer, et al. (J. Amer.Chem. Soc. 62, 1723–1732 (1940)). Namely, the evaluation can beperformed by measuring the amount of moisture absorption at eachrelative humidity by an indirect method using a saturated solution ofsalts, and by using the BET equation. In the preparation of the chitinsuitable for the extracellular matrix according to the presentinvention, the internal surface area of the material chitin shouldpreferably be 180 m²/g or more.

Thus, by evaluating the affinity for water of the chitin extracted froman insect, it is possible to use only chitin that has been evaluated tohave a high affinity for water as the extracellular matrix for thepresent invention. In this sense, the water-soluble chitin prepared fromthe pupa of an insect, particularly from silkworm pupa exuviae issuperior. The chitin prepared from silkworm pupa exuviae has a largeinternal surface area, with more water molecule adsorption sites thanother silkworm-derived cuticle. The chitin also has a high heat ofadsorption, and its affinity for water is greater than Tensan (Japanesesilkworm moth), Sakusan (Chinese silkworm moth), and cicadae. Further,the chitin derived from silkworm pupa exuviae has the same level ofaffinity for water as that of chitin derived from crabs and lobsters.One example of the silkworm that can be used in the present invention isa polyphagous silkworm race (adapted to new low cost artificial diet(LPY) lacking mulberry leaf powder) (N601×N602)×(C602×C603) (nicknamed“shin-asagiri”).

The thus obtained chitin is deacetylated in the following manner tothereby obtain the insect-derived water-soluble chitin according to thepresent invention.

Deacetylation means the removal of an acetyl group from chitin.Deacetylation can be performed by treating the above-extractedwater-soluble chitin with alkali or acid, preferably concentrated alkalior acid. This treatment may involve heating. The treatment may also beperformed by a reaction such as Clemmenssen reduction. However, bytreating with a concentrated alkaline solution to take advantage of thefact that chitin dissolves in concentrated alkaline, the deacetylationcan be performed in a homogeneous system.

The chitin according to the present invention is used afterdeacetylation, i.e., after removal of the N-acetyl group. Whileconventional chitin derived from crustaceans has been subjected to avariety of chemical modifications such as, e.g., acylation, tosylation,and carboxylmethylation, the water-soluble chitin according to theinvention may only need be subjected to deacetylation. Preferably,deacetylation is performed in a homogeneous system to take advantage ofthe chitin's solubility in a concentrated alkaline solution. Since in areaction in a heterogeneous system, N-acetyl groups are preferentiallyremoved from the chitin molecule surface and amorphous portion, therearises a deviation in the distribution of N-acetyl groups. On the otherhand, deacetylation proceeds in a random manner in a homogeneous system,so that remaining N-acetyl groups are distributed in a dispersed manner.As a result, hydrogen bonding between chitin molecules weakens, andwater-solubility develops. Specifically, it is reported that partiallydeacetylated chitin with a deacetylation degree in the range of from 40to 60% dissolves in water (Kramer. K. J et al., Insect Biochem., 14(3),pp. 293–298 (1984)).

Thus, deacetylation is performed in a homogeneous system with the use ofa chitin-added, high-viscosity alkaline solution, with the reactionconditions being set such that the degree of deacetylation is in therange of from 45 to 55%. Partially deacetylated chitin may be identifiedbased on an FT-IR. (Fourier transform infrared spectroscopy) spectrum.The degree of deacetylation may be easily measured by IR (infrared)spectroscopy. As chitin is turned into partially deacetylated chitin bydeacetylation, absorption by amide groups specific to the N-acetyl groupdecreases. Accordingly, the degree of deacetylation can be inferredbased on the IR spectrum. Specifically, the deacetylation degree may bemeasured based on the ratio of A₁₅₆₀/A₁₀₇₀, where the amide II band of1560 cm⁻¹ in the IR spectrum is used as a characteristic band forquantitation, and a band of 1070 or 1039 cm⁻¹ is used as an internalstandard. The degree of deacetylation may also be determined by using ananalytical curve which has been prepared in advance with reference to adeacetylated chitin sample as a control.

Thus, the insect-derived water-soluble chitin that has been deacetylatedas the sole chemical modification according to the present invention hashigh affinity for water, with a deacetylation degree in the range offrom 40 to 60%, preferably from 45 to 50%.

The thus obtained insect-derived water-soluble chitin that has beendeacetylated as the sole chemical modification comprises 40–60% chitin,and the remaining 40–60% has a chitosan structure. In other words, theinsect-derived water-soluble chitin that has been subjected todeacetylation as the sole chemical modification has high affinity forwater, of which 40–60% is chitin and the remaining 40–60% is chitosan.Since the cell membrane surface is negatively charged, the cells attachto the positively charged amino groups of the chitosan and can divideand multiply. This water-soluble chitin can be used as the extracellularmatrix.

The water-soluble, partially deacetylated chitin has about six timesmore water adsorption than prior to treatment and exhibits a highhygroscopicity, making it also suitable for use in food and cosmeticsfields as a moisture retention or absorption agent.

4. Use of the Extracellular Matrix According to the Invention

The water-soluble chitin according to the present invention is dissolvedin water at a concentration of 0.001 to 1% (W/V). The solution is thenpoured in the cell culture vessel in such a manner as to spread over thecell attached surface, and air-dried, thereby attaching thewater-soluble chitin to the surface of the culture vessel and thuscoating the same. A good coating can be obtained by, for example,putting 0.5 mL of the aqueous solution of chitin per 200 mm² of the cellculture flask surface into the vessel and air-drying.

Accordingly, the scope of the present invention includes the culturevessels including, e.g., a culture flask, petri dish, and plate whoseinner surface is partially or entirely coated by the extracellularmatrix according to the invention.

By culturing an insect cell in the cell culture vessel coated with theextracellular matrix according to the invention, the cell can attach tothe culture vessel and successfully multiply and divide.

5. Preparation of an Insect Culture Cell Line Using the Insect CellPrimary Culture Medium and the Deacetylated, Insect-derived ChitinAccording to the Invention

By using a vessel coated with the deacetylated, insect-derived chitinaccording to the invention, an insect culture cell line can beefficiently prepared on an insect cell primary culture medium accordingto the invention.

A tissue is extracted from an insect in an aseptic manner, and thetissue is put into a vessel coated with a deacetylated, insect-derivedchitin according to the invention as the extracellular matrix. Theinsect cell primary culture medium according to the invention to which afetal bovine serum has been added is also put into the vessel, andculturing is initiated. At this time, the vessel coated with theextracellular matrix should preferably be washed once with a sterilephysiological salt solution. An insect of any order may be used, but aninsect of the orders Lepidoptera, Diptera, Coleoptera, or Hemiptera, forexample, is particularly suitable. Tissue may be of any kind, butparticularly suitable are an embryonic tissue, fat body tissue,reproductive tissue such as that of testicles or ovary, digestivetissue, nervous tissue, and muscle tissue. Culturing is continued untilthe cells emerging from the tissue section onto the culture vesselsurface divide repeatedly and reach sufficient numbers. Cultureconditions may be those ordinarily used for insect cell culture. Duringthis period, half of the medium in the culture vessel is exchanged withfresh medium at appropriate intervals, such as every one or two weeks,so that cell transmigration from the tissue can be activated. After celltransmigration begins and a number of cell colonies are formed on theculture vessel bottom and have grown large, the content of fetal bovineserum in the fetal bovine serum-added primary culture medium isgradually decreased. For example, 30% fetal bovine serum may be added atthe start of culture, and 20% fetal bovine serum may be added when thecell populations have grown large. Thereafter, half of the medium isreplaced with a known subculture medium such as MGM-464 medium, IPL-41medium, Grace's medium, EXCELL 400-line medium, Sf900II medium, andSchneider medium, etc., and subculture is carried out. By replacing halfof the medium at appropriate intervals, the cells can be eventuallycultured on the target subculture medium.

In the following, the details of the process for the production of acell line derived from an intestinal tissue of silkworm will bedescribed by way of example.

1. Wash the body surface of an aseptically grown silkworm three timeswith a sterile Carlson's solution (penicillin 100000 U/100 mL, 0.05%gentamicin, and 0.05% antiformin have been added).

2. Immerse the silkworm in the last Carlson's solution for 1–2 min toterminate the movement of the silkworm.

3. Cut off the tail portion of the silkworm. Strip the thoracic skin bya pair of tweezers. Grasp the head part with the tweezers, and pull outthe intestinal tissue. Immerse the intestinal tissue in a Carlson'ssolution in a petri dish.

4. Collect the Peritrophic membrane with the tweezers.

5. Put it in an Eppendorf tube.

6. Put collagenase I liquid in the Eppendorf tube (4000 U/0.5 g tissueweight/collagenase mL)

7. Allow to stand at 27° C. for 2 hours.

8. Flush-centrifuge in a small desktop centrifuge (Chibitan™) for tensec.

9. Dispose of the supernatant liquid and wash twice with a balanced saltsolution.

10. Pipette strongly.

11. Transfer to a 15 mL disposable centrifugal tube, and centrifuge at500–800 rpm for 1 min.

12. Dispose of the supernatant liquid. Add a balanced salt solutionagain, centrifuge at 1000 rpm for 1 min, and dispose of the supernatantliquid. There is obtained a cell aggregate in the form of a pellet.

13. Put in a MX30 medium, transfer to a 24-multiwell plate and culture.Coat the culture surface of the plate in advance with a 0.01%(W/V)-concentration chitin (particularly in the cases of primary cultureof a blood corpuscle cell system, ovary, testis or embryo tissue).

14. Culture at 25° C.

15. Replace half of the medium (0.5–0.7 mL) every two weeks.

EXAMPLES

The present invention will hereafter be described in detail by way ofExamples. These Examples, however, should not be taken as limiting thetechnical scope of the present invention.

Example 1 Preparation of a Insect Cell Primary Culture Medium

The following substances were dissolved in purified water in amountsindicated below per 1 L of the medium to prepare a medium, which wasused as the MX medium.

(mg/1000 mL medium)

Composition of mineral salt mixtures NaH₂PO₄.2H₂O 507 NaHCO₃ 300 KCl1720 CaCl₂.2H₂O 750 CuCl₂.2H₂O 0.1 CoCl₂.6H₂O 0.03 FeSO₄.7H₂O 0.28MgCl₂.4H₂O 1140 MgSO₄.7H₂O 3269 MnCl₂.4H₂O 0.01 NaCl 1425 NaH₂PO₄.H₂O580 (NH₄)₆(Mo₇O₂₄.4H₂O) 0.02 ZnCl₂ 0.02 Composition of sugars D-glucose2917 Fructose 20.9 Sucrose 11865 Malic acid 306 α-ketoglutaric acid 169Succinic acid 27.4 Fumaric acid 25.2 Maltose 500 Composition of aminoacid mixtures L-α-alanine 131.5 β-alanine 234 L-arginine.HCl 692L-asparagine 797 L-asparaginic acid 797 L-cystine 10.5 L-glutamic acid1000 L-glutamine 750 Glycine 371 L-histidine 1142 L-isoleucine 396L-leucine 157 L-cystine.2Na 60 L-hydroxyproline 400 L-lysine.HCl 610L-methionine 521 L-phenylalanine 562 L-proline 396 DL-serine 559L-threonine 173 L-tryptophan 91.5 L-tyrosine 21 L-tyrosine.2Na 180L-valine 292 L-histidine 1142 Composition of vitamin mixtures Biotin0.123 D-calcium pantothenate 0.089 Choline chloride 10.85 Folic acid0.125 I-inositol 0.285 Nicotinic acid 0.165 Pyridoxine.HCl 0.285Riboflavin 0.125 Thiamine.HCl 0.125 Vitamin B₁₂ 0.12 Para-aminobenzoicacid 0.245 Composition of protein extracts Lactalbumin hydrolysate 1500TC-yeastolate 1500 Tryptose phosphate broth 1500 Fetuin 10 Cytochrome c50 Inosine 100 Bovine serum albumin V 5000 Viscosity-supplementing agentPolyvinylpyrrolidone K-90 250

To this prepared medium was further added fetal bovine serum (FBS) to20% (thereby making MX20 medium) and to 30% (thereby making MX30medium).

The thus prepared media were adjusted to pH 6.3 by potassium hydroxide.The prepared media were sterilized and stored in a Stericup™ (Millipore,SCGV05012) filtering and sterilizing vessel.

Example 2 Preparation of a Deacetylated Insect-derived Water-SolubleChitin

1. Preparation of Water-Soluble Chitin from Silkworm Pupa Exuviae

(i) Preparation Process

Five grams of the pupa exuviae of a polyphagous silkworm race (adaptedto new low cost artificial diet (LPY) lacking mulberry leaf powder)(N601×N602)×(C602×C603) (nicknamed “shin-asagiri”) was put in 300 mL of1 N aqueous solution of hydrochloric acid, and treated at 100° C. for 20min in an environment filled with nitrogen gas. Thereafter, the pupaexuvia was washed with warm water and distilled water until it wasneutral, and then vacuum-dried. The dried pupa exuvia was then immersedin 300 mL of 1 N solution of sodium hydroxide, and stirred at 80° C. for36 hours, to thereby remove the protein in the pupa exuvia. As a result,0.9 g of chitin was obtained.

(ii) Evaluation of Affinity for Water

Hygroscopicity of the obtained chitin was analyzed according to themethod of Yano, Bull, Mellon, Ashpole et al. by measuring the moistureuptake at each relative humidity by an indirect method involving asaturated solution of salts, while applying the BET equation.

As a result, since the obtained chitin had a large internal surface areaand a high heat of adsorption, the affinity of the chitin for water wasevaluated to be high.

(iii) Deacetylation of Chitin

Three grams of the above chitin obtained from silkworm pupa exuvia wasadded to a 40% solution of sodium hydroxide. The solution was allowed tostand at 25° C. for 70 hours, to thereby deacetylate the chitin in ahomogeneous solution system. There was synthesized partiallydeacetylated chitin in a 74% yield. When the obtained deacetylatedchitin was analyzed by IR spectroscopy, the degree of deacetylation was45–48%. This partially deacetylated chitin exhibited water solubility atroom temperature. It was also shown that the water adsorption of thepartially deacetylated chitin at the saturated vapor pressure was 6.3times as much as prior to the treatment, thus indicating a highhygroscopicity.

This deacetylated chitin was used as the extracellular matrix.

Example 3 Production of a Cell Culture Line

Zero-point-five milliliter of a 0.01–0.1% aqueous solution of the abovedeacetylated, water-soluble chitin derived from silkworm pupa exuvia waspoured into individual culturing wells of a culturing multiwell plate(Sumitomo Bakelite, MS-8024R) such that the solution reached the entirebottom surface of the plate. The solution was then air-dried at roomtemperature to thereby evaporate the liquid component. Thereafter, theculture surface was washed once with a sterile physiological saltsolution.

The fat body was extracted from the silkworm larva in an aseptic manner,and several tens of mg of the fat body was put in the wells of themultiwell plate, in which 1.5 mL (per 200 square mm of the bottom areaof the wells) of the MX30 medium treated and prepared according to themethod of Example 1 had been put. Half of the medium was replaced every14 days. After 30 days, transmigration of cells from the tissue becameactive, and, after two to three months, a number of cell populationsformed on the culture surface and grew larger. Microscopic observationrevealed the cells to be firmly attached to the flask bottom surface.Thereafter, half of the medium was replaced with MX20 medium. Further,half of MX20 medium was replaced every 14 days. After severalreplacements and confirming a sufficient growth of the cells, the mediumwas strongly blown onto the cell populations with the use of a culturingPasteur pipette, so that the cells could be floated from the culturesurface and transplanted into a new culture flask (Falcon,traditional-type flask No. 3018). The procedure of culturing andtransplanting into a new flask after cell multiplication was repeated.During this period, the medium was gradually replaced with a 10%FBS-added medium for subculture and, thereafter, the cells weresubcultured as an established culture cell line repeatedly to maintainthe cells.

Thus, the medium according to the present invention for the primaryculture of insect cells is a novel insect cell primary culture medium atleast comprising, as protein extracts, lactalbumin hydrolysate,yeastolate, and tryptose phosphate broth, and, as aviscosity-supplementing agent, polyvinylpyrrolidone. By using themedium, a culture cell line can be established from various tissues ofinsect efficiently in a short period of time.

The insect-derived water-soluble chitin which is deacetylated as thesole chemical modification according to the present invention can beused for coating a cell culture vessel. The coating enables cells toadhere to the culture vessel surface and efficiently grow and divide.

By performing primary culture with the insect cell primary culturemedium and the insect-derived water-soluble chitin which is deacetylatedas the sole chemical modification according to the present invention, acell line can be established in a shorter period of time and moreefficiently.

1. An insect cell culture vessel coated with a silkworm-derivedwater-soluble chitin having a degree of deacetylation of 45 to 55%,which is prepared by dissolving a silkworm-derived chitin in aconcentrated alkali water solution and allowing the resultant highviscosity alkaline chitin water solution to stand.
 2. A matrix for cellculture, comprising a silkworm-derived water-soluble chitin having adegree of deacetylation of 45 to 55%, which is prepared by dissolving asilkworm-derived chitin in a concentrated alkali water solution andallowing the resultant high viscosity alkaline chitin water solution tostand.
 3. The matrix for cell culture of claim 2 comprising 0.001% to 1%of said silkworm-derived water-soluble chitin.
 4. A process of preparingan insect culture cell line comprising culturing cells derived from aninsect in a cell culture vessel coated with the matrix for cell cultureof claims 2 or 3 using an insect primary culture medium comprisinglactalbumin hydrolysate, yeastolate and tryptose phosphate broth asprotein extracts and polyvinylpyrrolidone as a viscosity-supplementingagent.
 5. The matrix for cell culture of claims 2 or 3, wherein saidmatrix is in the form of a coating.
 6. The matrix for cell culture ofclaims 2 or 3 wherein said matrix is in the form of a coating obtainedby air drying an aqueous solution of said silkworm water-soluble chitin.